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目的:构建鸡贫血病毒(chicken anemia virus,CAV)VP3基因和肿瘤坏死因子相关凋亡诱导配体(TNF-related apopto-sis-inducing ligand,TRAIL)基因的真核表达载体,并观察其对胃癌细胞生长的影响情况。方法:采用分子克隆技术,将VP3基因插入真核表达载体pBud-TRAIL中,构建获得质粒pBud-TRAIL-VP3,采用电转化法将该重组质粒转入减毒沙门菌SL7207,再直接转染胃癌细胞株SGC-7901。48h后采用RT-PCR检测VP3基因表达状况并在荧光显微镜下观察细胞内有无绿色荧光蛋白(green fluorescent protein,GFP)表达。MTT法检测重组菌株对SGC-7901细胞生长状况的影响。AO/EB荧光染色检测其对肿瘤细胞凋亡率的影响。透射电镜观察胃癌细胞形态改变。结果:获得VP3基因正确插入的真核表达载体;重组质粒经减毒沙门菌介导转染胃癌细胞后,RT-PCR检测到VP3基因存在;荧光显微镜下观察到在转染细胞内有GFP表达;转染48h后可见到TRAIL和VP3对胃癌细胞生长有明显抑制作用(P<0.05),荧光染色可见胃癌细胞有凋亡现象,pBud-TRAIL-VP3能够明显提高胃癌细胞的凋亡率(P<0.05),透射电镜观察到转染细胞发生凋亡形态变化。结论:减毒沙门菌可将外源基因VP3和TRAIL基因导入胃癌细胞并进行表达,TRAIL和VP3对胃癌细胞的生长起协同抑制作用并能促进胃癌细胞的凋亡。
Objective: To construct eukaryotic expression vector of VP3 gene of chicken anemia virus (CAV) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) The impact of cell growth. METHODS: The VP3 gene was inserted into the eukaryotic expression vector pBud-TRAIL by molecular cloning. The plasmid pBud-TRAIL-VP3 was constructed. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207 by electroporation and then directly transfected into gastric cancer The expression of VP3 gene was detected by RT-PCR after SGC-7901.48h and the expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. The effect of recombinant strain on the growth of SGC-7901 cells was detected by MTT assay. AO / EB fluorescence staining to detect the impact of apoptosis rate of tumor cells. Transmission electron microscope observation of gastric cancer cell morphology changes. Results: VP3 gene was inserted into the eukaryotic expression vector correctly. The recombinant plasmid was transfected into gastric cancer cells by attenuated Salmonella, and VP3 gene was detected by RT-PCR. GFP expression was observed under the fluorescence microscope (P <0.05). After 48h transfection, TRAIL and VP3 could significantly inhibit the growth of gastric cancer cells (P <0.05). The apoptosis of gastric cancer cells was observed by fluorescence staining. The apoptosis rate of gastric cancer cells was significantly increased by pBud-TRAIL-VP3 <0.05). The morphological changes of apoptotic cells were observed by transmission electron microscopy. CONCLUSION: The attenuated Salmonella can transduce the exogenous gene VP3 and TRAIL gene into gastric cancer cells and express them. TRAIL and VP3 can inhibit the growth of gastric cancer cells and promote the apoptosis of gastric cancer cells.